A Micro-diffusion Technique for Visualizing Nucleic Acid Molecules
Contour lengths, molecular weights, and topological parameters of single molecules of nucleic acids can be studied accurately by electron microscopy. However, specialized equipment and highly purified samples of DNA or RNA in an amount of 10-100 μgm have been required. We have developed a simple micro-diffusion technique which uses less than 0.01 (μgm of nucleic acid. Droplets containing the nucleic acid are placed on a clean teflon sheet. Cytochrome C powder is added by needle to form a monofilm. After 20-30 minutes diffusion time, there is usually enough material attached for the film to be picked up on a suitable specimen grid, shadow-cast or stained, and examined in the electron microscope. Each droplet provides material for a single observation, but experiments may be carried out contiguously on the sheet.The method has been developed for viral nucleic acids, but can also be applied to solutions of virus particles where the genomes can be released by osmotic shock or by other physical procedures. Less than 1010 particles per ml are required for this modification. Examples of nucleic acids liberated from papovaviruses, SV40, rabbit papilloma, human papilloma, and from adeno-associated satellite virus will be presented. Viral nucleic acids (DNAs from SV40 and adenovirus, RNA from bacteriophage R17) prepared by conventional techniques will also be shown.